Type 2 diabetes affects over 29 million Americans (12.3% of adults over the age of 20). The prevalence of diabetes in Veterans is approximately double that in the general population and continues to rise. Dietary interventions to control or prevent type 2 diabetes could be highly effective and affordable, but reduced calorie diets have proven to be unsustainable over the long term. Diet plans without a decrease in caloric consumption that instead alter the level of specific macronutrients have therefore been seen as more sustainable by both researchers and the public. Intriguingly, recent studies in mice and humans have found that low dietary protein intake is positively associated with health and insulin sensitivity; however, the physiological and molecular mechanisms by which a low protein diet promotes metabolic health is not fully understood. We recently determined that decreased dietary intake of the three branched chain amino acids (BCAAs; leucine, isoleucine, and valine) recapitulates many metabolic benefits of a low protein diet, promoting leanness and glycemic control even in mice with pre-existing diet-induced obesity and type 2 diabetes. Our preliminary data suggests that a low BCAA diet promotes glucose tolerance in part by reducing hepatic gluconeogenesis and increasing hepatic insulin sensitivity, an effect that may be mediated by the AA-sensing kinase, GCN2. The central hypothesis examined here is that reducing levels of one or more dietary BCAAs alters signaling through the amino acid-sensing kinase GCN2 or other mediators, leading to favorable physiological changes that promote metabolic health in both inbred and genetically heterogeneous mice as well as in humans. Our long-term goal is to gain mechanistic insight into how reducing dietary BCAAs promotes metabolic health, identifying new points of intervention that may be targeted with pharmaceutical interventions or dietary strategies and enabling better therapeutic options to prevent and treat obesity and type 2 diabetes in Veterans. In this proposal, we will determine the specific contribution of each of the three BCAAs on metabolic health in the context of a Western diet, performing metabolic phenotyping and quantitatively determining the effect of altered BCAAs on the liver in vivo using hyperinsulinemic-euglycemic clamps and ex vivo in primary hepatocytes. We will test if our findings are applicable beyond inbred C57BL/6J mice by determining if reducing BCAAs promotes metabolic health in genetically heterogeneous mice. Finally, we will undertake a two-pronged approach to gain mechanistic insight into the molecular mechanisms by which reduced BCAAs promote metabolic health. First, we will test the role of GCN2 using a genetic mouse model lacking hepatic Gcn2. Second, we will identify candidate molecular mediators by proteomic and metabolomics profiling of the livers of mice fed a reduced BCAA diet, and test the role of these candidate mediators in the regulation of hepatocyte glucose metabolism in a cell culture system. The innovative preclinical studies described in this proposal will significantly advance our understanding of how specific dietary BCAAs regulate metabolic health in mice, filling an important gap in present knowledge. While the present studies will be conducted in mice, all of the questions are directly relevant to our current and ongoing efforts to translate our preliminary findings into humans, with the goal of improving preventative and therapeutic options for Veterans who are obese or who have type 2 diabetes.